Introduction

In this vignette we’ll focus on one of the more complicated features of scLANE: the usage of GEE models to identify dynamic genes when cells are grouped by some subject identity. The robust variance estimation available in GEE models allows us to make decisions about which genes are actually dynamic over pseudotime / latent time while accounting for intra-subject correlations between observations. The downsides are that the benefits of GEE models can be a little more difficult to explain to collaborators, and they take a little bit longer than GLMs to estimate. A refresher on what GEEs are and why they’re useful can be found here.

Libraries

library(dplyr)      # data manipulation
library(scran)      # single cell tools 
library(scLANE)     # differential expression over pseudotime
library(scater)     # single cell tools 
library(slingshot)  # pseudotime estimation

Data

First we’ll load in the lung tumor data from Zilionis et al (2019), and subset to only include tumor neutrophils. The authors found 5 continuous tumor neutrophil subsets in their analysis, and we’re interested in determining which genes drive subset-to-subset differentiation. The data were collected from 7 different patients, and thus the usage of GEE models is warranted in order to properly estimate within-patient gene expression correlations.

lung <- scRNAseq::ZilionisLungData(which = "human", filter = TRUE)
lung <- lung[, stringr::str_detect(lung$`Major cell type`, "tNeutrophils")]

Preprocessing

First we’ll preprocess the data in the typical way. We see that the neutrophils fall into 5 clusters, and that there is one odd outlier group of cells near the top of the UMAP plot. In addition, there is a decent amount of intra- and inter-patient variability. The cluster labels are not solely reflective of the cell subtypes, and seem to be driven by both patient identity as well as cell subtype.

lung <- logNormCounts(lung)
var_decomp <- modelGeneVar(lung)
top2k_hvgs <- getTopHVGs(var_decomp, n = 2000)
lung <- runPCA(lung, subset_row = top2k_hvgs)
reducedDim(lung, "PCAsub") <- reducedDim(lung, "PCA")[, 1:10, drop = FALSE]
lung <- runUMAP(lung, dimred = "PCAsub", n_dimred = 1:10)
g <- buildSNNGraph(lung, use.dimred = "PCAsub", k = 40)
clusters <- igraph::cluster_louvain(graph = g)$membership
colLabels(lung) <- factor(clusters)
plotUMAP(lung, colour_by = "label")

plotUMAP(lung, colour_by = "Patient")

plotUMAP(lung, colour_by = "Minor subset")

Estimating Pseudotime

We’ll use slingshot to estimate a pseudotime value for each cell. In this case, we see only one pseudotime lineage.

scl_lineage <- getLineages(reducedDim(lung, "PCAsub"), clusterLabels = clusters)
scl_crv <- getCurves(scl_lineage)
scl_cell_weights <- slingCurveWeights(scl_crv)
scl_pt <- slingPseudotime(scl_crv)
pt_df <- as.data.frame(scl_pt) %>% 
         mutate(across(everything(), function(x) x / max(x, na.rm = TRUE)))

Running scLANE

Global Test

We’re going to run scLANE solely on the top 10 most highly variable genes. This is for computational quickness, as otherwise the models would take a couple hours to run & this is a tutorial. Note that we need to provide a (sorted) vector of subject IDs as well as a working correlation structure. In this case we opt for the exchangeable (AKA compound symmetry) structure, as it stands to reason that within-subject cells will have similar correlations.

lung_counts <- t(lung@assays@data$counts)
lung_counts <- as.matrix(lung_counts[, which(colSums(lung_counts) > 0)])
gene_stats <- testDynamic(expr.mat = lung_counts[, colnames(lung_counts) %in% top2k_hvgs[1:10]], 
                          genes = top2k_hvgs[1:10], 
                          pt = pt_df, 
                          is.gee = TRUE, 
                          id.vec = lung$Patient, 
                          cor.structure = "exchangeable", 
                          parallel.exec = TRUE, 
                          n.cores = 4, 
                          track.time = TRUE)
[1] "testDynamic evaluated 10 genes with 1 lineages apiece in 8.388 mins"

Let’s check out the results of the global differential expression test.

getResultsDE(gene_stats) %>% 
  select(-contains("LogLik"), -contains("Dev")) %>%  # not relevant to GEEs
  kableExtra::kbl(digits = 5, 
                  align = "c", 
                  booktabs = TRUE, 
                  caption = "Lineage-level dynamic test", 
                  col.names = c("Gene", "Lineage", "Test Stat.", "P-value", "Test Stat. Type", 
                                "Model Status", "Adj. P-value", "Gene Dynamic over Lineage", "Gene Dynamic")) %>% 
  kableExtra::kable_paper("hover", full_width = FALSE)
Lineage-level dynamic test
Gene Lineage Test Stat. P-value Test Stat. Type Model Status Adj. P-value Gene Dynamic over Lineage Gene Dynamic
CXCL8 A 2102.214 0 Wald MARGE & null model OK 0 1 1
IL1RN A 1388.697 0 Wald MARGE & null model OK 0 1 1
IL1B A 643.460 0 Wald MARGE & null model OK 0 1 1
RGS2 A 20333.466 0 Wald MARGE & null model OK 0 1 1
S100A8 A 139.937 0 Wald MARGE & null model OK 0 1 1
HSPA1A A 11437468.161 0 Wald MARGE & null model OK 0 1 1
PI3 A 0.000 1 Wald MARGE & null model OK 1 0 0
NFKBIA A NA NA Wald MARGE model error, null model OK NA 0 0
ACTB A NA NA Wald MARGE model error, null model OK NA 0 0
CCL4 A NA NA Wald MARGE model error, null model OK NA 0 0

Slope Test

Next we can run the slope test to identify over which pseudotime intervals gene expression changes significantly.

testSlope(test.dyn.results = gene_stats) %>% 
  kableExtra::kbl(digits = 5, 
                  align = "c", 
                  booktabs = TRUE, 
                  caption = "Slope-level dynamic test", 
                  col.names = c("Gene", "Lineage", "Breakpoint", "Rounded Breakpoint", 
                                "Direction", "P-value", "Notes", "Adj. P-value", 
                                "Gene Dynamic over Lineage-Slope", "Gene Dynamic over Lineage", 
                                "Gene Dynamic")) %>% 
  kableExtra::kable_paper("hover", full_width = FALSE)
Slope-level dynamic test
Gene Lineage Breakpoint Rounded Breakpoint Direction P-value Notes Adj. P-value Gene Dynamic over Lineage-Slope Gene Dynamic over Lineage Gene Dynamic
ACTB 1 NA NA NA NA MARGE model error, null model OK NA 0 0 0
CCL4 1 NA NA NA NA MARGE model error, null model OK NA 0 0 0
CXCL8 A 0.49615 0.4962 Right 0.00000 NA 0.00001 1 1 1
CXCL8 A 0.49805 0.4980 Right 0.00000 NA 0.00001 1 1 1
HSPA1A A 0.00576 0.0058 Right 0.00000 NA 0.00000 1 1 1
HSPA1A A 0.76835 0.7683 Right 0.00000 NA 0.00000 1 1 1
IL1B A 0.27310 0.2731 Right 0.12353 NA 1.00000 0 0 0
IL1B A 0.27429 0.2743 Right 0.13126 NA 1.00000 0 0 0
IL1RN A 0.00576 0.0058 Right 0.00000 NA 0.00000 1 1 1
IL1RN A 0.28645 0.2865 Left 0.00000 NA 0.00000 1 1 1
NFKBIA 1 NA NA NA NA MARGE model error, null model OK NA 0 0 0
PI3 A NA NA NA NA No non-intercept coefficients NA 0 0 0
RGS2 A 0.00576 0.0058 Right 0.00000 NA 0.00000 1 1 1
S100A8 A 0.18537 0.1854 Left 0.01513 NA 0.16639 0 1 1
S100A8 A 0.18667 0.1867 Right 0.00000 NA 0.00000 1 1 1

Model Visualization

Lastly, we can plot the fitted model for a couple of the genes.

plotModels(test.dyn.res = gene_stats, 
           gene = "CXCL8", 
           pt = pt_df, 
           gene.counts = lung_counts, 
           is.gee = TRUE, 
           id.vec = lung$Patient, 
           cor.structure = "exchangeable")

plotModels(test.dyn.res = gene_stats, 
           gene = "RGS2", 
           pt = pt_df, 
           gene.counts = lung_counts, 
           is.gee = TRUE, 
           id.vec = lung$Patient, 
           cor.structure = "exchangeable")

Session Info

sessioninfo::session_info()
─ Session info ───────────────────────────────────────────────────────────────
 setting  value                       
 version  R version 4.0.4 (2021-02-15)
 os       macOS Big Sur 10.16         
 system   x86_64, darwin17.0          
 ui       X11                         
 language (EN)                        
 collate  en_US.UTF-8                 
 ctype    en_US.UTF-8                 
 tz       America/New_York            
 date     2022-04-30                  

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[1] /Library/Frameworks/R.framework/Versions/4.0/Resources/library
---
title: "Identifying Dynamic Genes with Generalized Estimating Equations in `scLANE`"
subtitle: "University of Florida - Dept. of Biostatistics - Bacher Group"
author: "Jack Leary"
date: "`r Sys.Date()`"
output:
  html_document:
    theme: yeti
    highlight: tango
    code_folding: show
    code_download: true
    toc: true
    toc_float:
      collpased: false
    df_print: kable
---

```{r setup, include=FALSE}
knitr::opts_chunk$set(echo = TRUE, 
                      comment = NA, 
                      message = FALSE, 
                      warning = FALSE, 
                      fig.align = "center")
set.seed(312)  # lucky seed
```

# Introduction 

In this vignette we'll focus on one of the more complicated features of `scLANE`: the usage of GEE models to identify dynamic genes when cells are grouped by some subject identity. The robust variance estimation available in GEE models allows us to make decisions about which genes are actually dynamic over pseudotime / latent time while accounting for intra-subject correlations between observations. The downsides are that the benefits of GEE models can be a little more difficult to explain to collaborators, and they take a little bit longer than GLMs to estimate. A refresher on what GEEs are and why they're useful can be found [here](https://www.publichealth.columbia.edu/research/population-health-methods/repeated-measures-analysis). 

# Libraries

```{r}
library(dplyr)      # data manipulation
library(scran)      # single cell tools 
library(scLANE)     # differential expression over pseudotime
library(scater)     # single cell tools 
library(slingshot)  # pseudotime estimation
```

# Data 

First we'll load in the lung tumor data from [Zilionis *et al* (2019)](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620049/), and subset to only include tumor neutrophils. The authors found 5 continuous tumor neutrophil subsets in their analysis, and we're interested in determining which genes drive subset-to-subset differentiation. The data were collected from 7 different patients, and thus the usage of GEE models is warranted in order to properly estimate within-patient gene expression correlations. 

```{r}
lung <- scRNAseq::ZilionisLungData(which = "human", filter = TRUE)
lung <- lung[, stringr::str_detect(lung$`Major cell type`, "tNeutrophils")]
```

## Preprocessing

First we'll preprocess the data in the typical way. We see that the neutrophils fall into 5 clusters, and that there is one odd outlier group of cells near the top of the UMAP plot. In addition, there is a decent amount of intra- and inter-patient variability. The cluster labels are not solely reflective of the cell subtypes, and seem to be driven by both patient identity as well as cell subtype. 

```{r}
lung <- logNormCounts(lung)
var_decomp <- modelGeneVar(lung)
top2k_hvgs <- getTopHVGs(var_decomp, n = 2000)
lung <- runPCA(lung, subset_row = top2k_hvgs)
reducedDim(lung, "PCAsub") <- reducedDim(lung, "PCA")[, 1:10, drop = FALSE]
lung <- runUMAP(lung, dimred = "PCAsub", n_dimred = 1:10)
g <- buildSNNGraph(lung, use.dimred = "PCAsub", k = 40)
clusters <- igraph::cluster_louvain(graph = g)$membership
colLabels(lung) <- factor(clusters)
plotUMAP(lung, colour_by = "label")
plotUMAP(lung, colour_by = "Patient")
plotUMAP(lung, colour_by = "Minor subset")
```

## Estimating Pseudotime

We'll use `slingshot` to estimate a pseudotime value for each cell. In this case, we see only one pseudotime lineage. 

```{r}
scl_lineage <- getLineages(reducedDim(lung, "PCAsub"), clusterLabels = clusters)
scl_crv <- getCurves(scl_lineage)
scl_cell_weights <- slingCurveWeights(scl_crv)
scl_pt <- slingPseudotime(scl_crv)
pt_df <- as.data.frame(scl_pt) %>% 
         mutate(across(everything(), function(x) x / max(x, na.rm = TRUE)))
```

# Running `scLANE` 

## Global Test

We're going to run `scLANE` solely on the top 10 most highly variable genes. This is for computational quickness, as otherwise the models would take a couple hours to run & this is a tutorial. Note that we need to provide a (sorted) vector of subject IDs as well as a working correlation structure. In this case we opt for the exchangeable (AKA compound symmetry) structure, as it stands to reason that within-subject cells will have similar correlations. 

```{r}
lung_counts <- t(lung@assays@data$counts)
lung_counts <- as.matrix(lung_counts[, which(colSums(lung_counts) > 0)])
gene_stats <- testDynamic(expr.mat = lung_counts[, colnames(lung_counts) %in% top2k_hvgs[1:10]], 
                          genes = top2k_hvgs[1:10], 
                          pt = pt_df, 
                          is.gee = TRUE, 
                          id.vec = lung$Patient, 
                          cor.structure = "exchangeable", 
                          parallel.exec = TRUE, 
                          n.cores = 4, 
                          track.time = TRUE)
```

Let's check out the results of the global differential expression test. 

```{r}
getResultsDE(gene_stats) %>% 
  select(-contains("LogLik"), -contains("Dev")) %>%  # not relevant to GEEs
  kableExtra::kbl(digits = 5, 
                  align = "c", 
                  booktabs = TRUE, 
                  caption = "Lineage-level dynamic test", 
                  col.names = c("Gene", "Lineage", "Test Stat.", "P-value", "Test Stat. Type", 
                                "Model Status", "Adj. P-value", "Gene Dynamic over Lineage", "Gene Dynamic")) %>% 
  kableExtra::kable_paper("hover", full_width = FALSE)
```

## Slope Test 

Next we can run the slope test to identify over which pseudotime intervals gene expression changes significantly. 

```{r}
testSlope(test.dyn.results = gene_stats) %>% 
  kableExtra::kbl(digits = 5, 
                  align = "c", 
                  booktabs = TRUE, 
                  caption = "Slope-level dynamic test", 
                  col.names = c("Gene", "Lineage", "Breakpoint", "Rounded Breakpoint", 
                                "Direction", "P-value", "Notes", "Adj. P-value", 
                                "Gene Dynamic over Lineage-Slope", "Gene Dynamic over Lineage", 
                                "Gene Dynamic")) %>% 
  kableExtra::kable_paper("hover", full_width = FALSE)
```

## Model Visualization 

Lastly, we can plot the fitted model for a couple of the genes. 

```{r, fig.width=8}
plotModels(test.dyn.res = gene_stats, 
           gene = "CXCL8", 
           pt = pt_df, 
           gene.counts = lung_counts, 
           is.gee = TRUE, 
           id.vec = lung$Patient, 
           cor.structure = "exchangeable")
plotModels(test.dyn.res = gene_stats, 
           gene = "RGS2", 
           pt = pt_df, 
           gene.counts = lung_counts, 
           is.gee = TRUE, 
           id.vec = lung$Patient, 
           cor.structure = "exchangeable")
```

# Session Info 

```{r}
sessioninfo::session_info()
```
